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- Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extractsPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Almeida, Sara; Coelho, Natacha; Quintas, Célia; Gaspar, Maria Nelma; Romano, AnabelaEvaluation of the antioxidant activity of the methanol, water and n-hexane extracts of Drosera intermedia, determined by the Folin-Ciocalteau (F-C), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays showed that the methanol extract had the highest antioxidant activity (F-C: 378.6 ± 31.5 μmolGAE/mgextract; TEAC: 332.2 ± 29.1 μmolTE/mgextract; ORAC: 64.7 ± 7.8 μmolTE/mgextract). Antimicrobial activity was tested against seven bacterial and eight yeast strains using the agar diffusion assay, followed by the determination of minimum inhibitory concentrations (MIC). All tested D. intermedia extracts demonstrated strong antimicrobial properties with a broad spectrum of activity. However, the n-hexane extract exhibited much greater activity than water and methanol extracts. The most susceptible microorganisms to the n-hexane extract were Staphylococcus epidermidis ATCC 12228 and Candida albicans YP0175, for which a MIC value of 13.0 μg/mL was scored.
- Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.)Publication . Teixeira, Monica; Coelho, N.; Olsson, M. E.; Brodelius, P. E.; Carvalho, Isabel Saraiva de; Brodelius, MariaTwo full-length cDNA clones of PoleFAD2 and one full-length cDNA clone of PoleFAD6, encoding omega-6 fatty acid desaturases, the key enzymes for the conversion of oleic into linoleic acid, were isolated from purslane (Portulaca oleracea L.) leaves and seeds. The deduced amino acid sequence of both isoforms of PoleFAD2 showed higher similarities to other microsomal omega-6 desaturases then to PoleFAD6 or other plastidial orthologues, and vice versa. Expression analysis by RT-PCR showed that all genes are expressed in all tissues of purslane tested, but higher levels of mRNA accumulation were detected in reproductive organs and cells that proliferate rapidly or store lipids. Wounding affected the levels of mRNA accumulation of both, FAD2 and FAD6 genes in purslane leaves, while chilling stress affected only FAD2 transcript level. The expression patterns observed reflect the discrete roles of these genes in membrane synthesis for cell division, thylakoid development, and lipid storage or in the biosynthetic pathway for the production of signaling molecules that influence plant development or defense.
- In vitro propagation of insectivorous plants for phytochemical purposesPublication . Coelho, Natacha; Romano, Anabela; Gonçalves, SandraThe aims of this research work were to establish in vitro propagation protocols for the insectivorous plants D. intermedia, D. rotundifolia and P. vulgaris, using seeds as explants, in order to produce plants for biocompound extraction and to replenish natural populations. The antioxidant capacity of the extracts prepared from in vitro cultures of these species was evaluated. The establishment of a simple protocol for in vitro propagation of D. intermedia is reported. Protocols for micropropagation of P. vulgaris and D. rotundifolia were initiated but still need to be improved. Cultures of all three species were initiated from shoots of in vitro produced seedlings. The effect of three MS medium concentrations, ¼MS, ½MS and MS, without PGR were tested in D. intermedia and P. vulgaris. The same media supplemented with 0.1 mg l-1 KIN were investigated in D. intermedia and ¼MS supplemented with 0.5 mg l-1 KIN or ZEA were investigated in P. vulgaris. In all media D. intermedia shoots rooted during the proliferation phase, presenting a multiplication and rooting percentage above 90%. The highest mean number of shoots was attained in ¼MS and ¼MS supplemented with 0.1 mg l-1 KIN. All the D. intermedia plants were successfully acclimatized to ex vitro conditions. The formation of roots also occurred during P. vulgaris proliferation phase, though with poorer results for both proliferation and rooting. Nevertheless, the best results were obtained in the media supplemented with cytokinins. In D. rotundifolia the media tested were ¼MS and MS. Multiplication percentage and mean number of shoots was higher in ¼MS medium and no rooting was observed. Antioxidant activity of D. intermedia, D. rotundifolia and P. lusitanica extracts was measured using three different methods: ORAC, F-C phenolics and TEAC. Methanol and standard maceration improved the efficiency of the antioxidant extraction procedure. For all tested methods P. lusitanica presented the highest antioxidant activity, representing a possible good source of antioxidants.